乙草胺对农田土壤细菌多样性的影响

在实验室条件下 ,将农田土壤分别用终浓度为 1 0mg、5 0mg和 1 0 0mg g土壤的乙草胺处理 4 0d后 ,检测可培养的异养细菌的多样性和细菌物种多样性。可培养的异养细菌的多样性依据在LB平板上的菌落形态来研究 ,细菌物种多样性则通过基因组DNA的提取 ,纯化 ,1 6SrDNA片段的扩增和变性梯度凝胶电泳 (DGGE)的分离来研究 ,香农多样性指数 (H) ,丰度 (S)和均匀度 (EH)等指标用于评价细菌多样性。实验结果表明 ,与对照土壤相比 ,处理土壤中上述两种类型的细菌多样性均降低 ,而且 ,不同处理浓度对土壤细菌多样性的影响也不同。将DGGE图谱中的条带回收并测序 ,结果显示乙草胺对土壤中的Proteobacteria的α Proteobacteria和γ Proteobacteria影响明显 ,表明乙草胺会在一定程度上改变土壤细菌多样性     

利用PCR-DGGE技术分析内蒙古西部地区土壤细菌的多样性

为明确内蒙古西部地区土壤细菌的多样性,利用聚合酶链反应-变性梯度凝胶电泳PCR-DGGE(polymerase chain reaction-denaturing gradient gel electrophoresis)技术对170份土壤样品中的细菌丰度和群落结构组成进行了分析,并研究了土壤细菌多样性与肥力参数的关系。结果表明:内蒙古西部地区土壤细菌的多样性是比较丰富的,丰富度指数处于4到60之间,香浓指数处于1.38到4.09之间。不同土壤类型,其细菌的多样性有明显差异。其中,新积土、棕钙土、栗钙土和灰钙土中的细菌多样性指数均高于其他类型土壤,而灰漠土的多样性指数最低。且土壤的不同利用方式也会对土壤细菌多样性有所影响,其中耕地土壤细菌多样性指数最高,而未利用土壤的多样性指数最低。细菌多样性与土壤肥力参数的相关性分析结果显示二者之间并无显著的相关性。内蒙古西部地区土壤中的优势种群包括Proteobacteria(变形菌门)、Bacteroidetes(拟杆菌门)、Firmicutes(厚壁菌门)、Actinobacteria(放线菌门)、Acidobacteria(酸杆菌门)、Gemmatimonadetes(芽单胞菌门)、Nitrospira(硝化螺旋菌门)。可为该地区的土壤生态系统治理与恢复及未来该地区特有微生物资源的开发利用、农业生产指导等提供一定的科学依据。     

乌梁素海湖滨湿地细菌群落结构多样性

【目的】了解乌梁素海湖滨湿地水陆过渡带细菌群落结构及多样性变化,探讨富营养化湖泊湿地基质条件对细菌群落结构的影响。【方法】应用变性梯度凝胶电泳(PCR-DGGE)技术,分析和比较了依陆向分布的4个水陆过渡带样点的湿地细菌群落结构多样性,采用典型对应分析(CCA)探讨了湿地基质因子对细菌多样性的影响。【结果】DGGE图谱显示依湖泊水体沉积物(S-1)→湖滨芦苇沼泽沉积物(S-2)→湖滨碱蓬盐化草甸土壤(S-3)→岸上白刺荒漠土壤(S-4),4个样点的条带数依次减少,对应菌群结构及多样性变化显著;多样性指数分析结果显示,Shannon-Wiener指数(H)、均匀度(E)、丰富度(S)以及Simpson指数(DS)均显示依陆向分布逐步下降的规律,即:S-1S-2S-3S-4。序列比对结果显示,沉积物及土壤细菌分属于变形菌门(78.6%)、酸杆菌门(7.1%)、拟杆菌门(14.3%)这3个细菌类群,优势菌门为变形菌门,而变形菌门又分为5个亚群,其中ε变形菌纲为优势亚群;CCA结果表明,图中各条带对应物种的分布受铵态氮、总氮、有机碳、水溶盐总量、氯离子以及钾离子影响最大。【结论】乌梁素海富营养化湖泊的水陆过渡带湿地细菌群落结构存在较大差异,富营养化相关基质因子对细菌多样性影响较大。这为研究富营养化湖泊湿地水陆过渡带的细菌结构多样性及空间异质性提供了科学依据。     

抗真菌转基因水稻秸秆降解对土壤细菌数量及多样性的影响

【背景】随着转基因作物的大面积种植,其潜在的环境安全性问题备受关注。转基因作物收获后,大部分残留物会重返土壤,可能对土壤微生物造成影响。【方法】通过室内模拟田间秸秆降解试验,采用平板计数法、表面荧光显微镜直接计数法（FDC）以及变性梯度凝胶电泳（DGGE）技术,分析了抗真菌转基因水稻秸秆降解对土壤细菌数量及多样性的影响。【结果】平板计数表明,在整个降解过程中,转基因与非转基因处理土壤可培养细菌数量的变化趋势有所差别,但差异不显著。FDC结果表明,转基因与非转基因处理土壤细菌总数差异不显著。DGGE指纹图谱显示,转基因与非转基因处理土壤样品之间的多样性指数、均匀度和丰富度均无显著差异。【结论与意义】抗真菌转基因水稻秸秆降解并未对土壤细菌数量和多样性产生显著影响。本研究为抗真菌转基因水稻的环境安全性评估提供了依据。     

PCR-DGGE技术用于湖泊沉积物中微生物群落结构多样性研究

采用PCR-DGGE分子指纹图谱技术比较南京市玄武湖、奠愁湖和太湖不同位置的表层沉积物微生物群落结构,研究结果表明,三湖泊沉积物微生物的16SrDNA的PCR扩增结果约为626bp,为16S rDNA V3～V5区特异性片段。玄武湖和莫愁湖表层沉积物中大约有20种优势菌群,且同一湖泊不同采样点DGGE图谱的差异性不大,细菌群落结构具有较高的相似性,而太湖样品DGGE条带的数目和位置表现出明显差异,且不同采样点图谱的差异性较大。三湖泊除具有特征性的微生物种属外,还分布约5个相同的细菌种群,可能与沉积物的理化性质和水生植被的影响相关。对DGGE图谱中7条主带进行回收、扩增和测序,结果显示其优势菌群具有不同的序列组成,其中5个序列与Genebank中已登录的细菌种群的同源性≥99％,2个序列的同源性为96％和93％,其中2个相似的细菌类群目前尚未获得纯培养。     

PCR-DGGE方法分析原油储层微生物群落结构及种群多样性

使用基于 16 S r DNA的 PCR- DGGE(变性梯度凝胶电泳 )图谱分析结合条带割胶回收 DNA进行序列分析 ,对新疆克拉玛依油田一中区注水井 (12 # 9- 11)和与该注水井相应的两个采油井 (12 # 9- 9S、13# 11- 8)井口样品微生物群落的多样性进行了比较并鉴定了部分群落成员。 DGGE图谱聚类分析表明注水井与两油井微生物群落的相似性分别为 30 %和 2 0 % ,而两油井间微生物群落结构的相似性为 5 4 %。DGGE图谱中优势条带序列分析表明注水井样品和油井样品中的优势菌群为未培养的环境微生物 ,它们与数据库中 α、γ、δ、ε变形杆菌 (Proteobacteria)和拟杆菌 (Bacteroidetes)有很近的亲缘关系。 DGGE与分子克隆相结合的分子生物学方法在研究微生物提高原油采收率 (MEOR)机理 ,以及指导 MEOR在油田生产中的应用有着重要的意义     

PCR-DGGE技术分析染整废水微生物群落多样性

旨在揭示水解酸化-生物接触氧化工艺处理染整废水过程中的微生物多样性.取初级沉淀池,水解酸化池,生物接触氧化池和二沉池的活性污泥,通过细胞裂解直接提取基因组DNA,以细菌通用引物进行16S rRNA基因V3区域PCR扩增,将PCR产物进行变性梯度凝胶电泳,获得微生物群落的DNA特征指纹图谱,并对条带进行统计分析和切胶测序,进行了同源性分析并建立了系统发育树.研究表明,整个水处理过程中含有丰富的微生物群落,其中初级沉淀池、水解酸化池、二沉池和生物接触氧化池的污泥样品分别测出36条带、42条带、30条带和29条带.不同区段微生物群落间相似度最高达68％,最低达42.4％,说明群落间演替明显,不同工艺区段既存在共同的微生物种属也存在特异微生物种属.     

茶园根际土壤细菌群落结构及多样性

【背景】茶园根际土壤的细菌群落结构与茶园生境土壤营养循环密切相关,其组成及多样性可以作为健康茶园的一个生物指标。【方法】采用PCR．变性梯度凝胶电泳（PCR—DGGE）分子指纹图谱技术,检测安溪铁观音种植区不同海拔茶园根际土壤样本的细菌群落结构,利用Shannon．Wiener多样性指数分析其多样性,采用非加权组平均法进行聚类分析得到其分布特征,利用蒙特卡罗检验和冗余分析分别揭示影响细菌群落分布的环境因子及细菌群落分布和环境变量之间的关系。【结果】茶园根际土壤细菌的DGGE结果显示,检测到的14种主要细菌中有11种细菌是不可培养的,3种细菌是可培养的,分别属于根瘤菌属、中华根瘤菌属和苍白杆菌属。聚类分析得到,同一海拔梯度茶园根际土壤细菌群落结构相似。Shan-non—Wiener多样性指数分析表明,400m海拔处茶园根际土壤细菌群落多样性最高。蒙特卡罗检验分析得到环境因子协同作用对茶园根际土壤细菌群落结构贡献率为59．6％。冗余分析显示,茶园根际土壤细菌群落结构与海拔密切相关。【结论与意义】茶园根际土壤细菌群落结构分布与海拔梯度密切相关,考虑不同海拔高度土壤细菌群落对茶园营养循环的影响,在铁观音的健康栽培和管理过程中具有重要意义。     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

云南腾冲热海两热泉菌藻席细菌多样性的研究

应用显微形态观察和变性梯度凝胶电泳(DGGE)对云南腾冲热海两热泉菌藻席的细菌多样性进行了比较分析.直接从环境样品中提取总DNA,用两套细菌通用引物进行PCR扩增,得到包含V8和V9高变区的16S rDNA片段,进行DGGE分析,结合形态观察,结果显示,热泉菌藻席中存在丰富的细菌多样性,且不同温度范围的菌藻席细菌组成差异显著.     

基于PCR-DGGE技术的红树林区微生物群落结构

【目的】为了解红树林沉积物中细菌的群落结构特征。【方法】应用PCR-DGGE技术对福建浮宫红树林的16个采样站位样品细菌的群落结构进行了研究。根据DGGE指纹图谱,对它们的遗传多样性进行了分析。【结果】各站位样品细菌多样性指数(H)、丰度(S)和均匀度(EH)均有所不同,这些差异与它们所处站位的不同有关,红树林区细菌多样性高于非红树林区细菌多样性。对不同站位细菌群落相似性分析,它们的相似性系数也存在一定的规律,同一断面的细菌群落结构相近性较高。对DGGE的优势条带序列分析,同源性最高的微生物分别属于变形菌门(Proteobacteria)、酸菌门(Acidobacteria)和绿菌门(Chlorobi),它们均为未培养微生物,分别来自于河口海岸沉积物。【结论】应用PCR-DGGE技术更能客观地反映红树林沉积物中真实的细菌群落结构信息。另外,研究也表明红树林区微生物多样性丰富,在红树林区研究开发未知微生物资源具有巨大的潜力。     

螺旋粉虱成虫体内细菌群落多样性的PCR-DGGE 和16S rRNA文库序列分析

为了解螺旋粉虱Aleurodicus dispersus体内细菌多样性和主要优势菌群结构, 用PCR-DGGE和16S rRNA文库对采自于海南省番石榴上螺旋粉虱雌、 雄成虫体内的细菌群落进行了分析。用PCR扩增体内细菌16S rRNA基因, 构建雌、 雄虫克隆文库; 再用限制性片段长度多态性（restriction fragment length polymorphism, RFLP）方法从文库中筛选不同16S rRNA基因图谱, 根据图谱对克隆子进行分型。从螺旋粉虱雌、 雄两个样品中共获得10 种分类操作单元(operational taxonomic unit, OTUs)。以16S rRNA基因为基础构建系统发育树, 系统发育分析表明, 螺旋粉虱雌、 雄成虫体内优势菌群主要为发酵菌属Zymobacter, 杀雄菌属Arsenophonus, 泛菌属Pantoea和假单胞菌属Pseudomonas。Candidatus Portiera aleyrodidarum和Arsenophonus sp.可能为其体内共生菌群, 在所有样品中均可稳定地检测到。这些微生物可能对螺旋粉虱生长发育、 繁殖和性比调控起到重要的协同作用。     

不同施氮方式和施氮量对马尾松和木荷幼苗根系土壤细菌群落的影响

作为影响全球变化的主要因子之一,氮沉降对生态系统生物地球化学循环有重要的影响。细菌作为土壤生态系统物质循环的关键参与者,是土壤生态系统变化的敏感指标,在氮沉降对生态系统影响过程中发挥不可忽视的作用。模拟2种施氮方式(SAN:土表施氮,LAN:叶面施氮)和3种施氮量(5.6、15.6、20.6 g N m^-2a^-1),运用PCR-DGGE技术,分析不同施氮方式和施氮量对两种盆栽植物马尾松(Pinus massoniana Lamb.)和木荷(Schima superba Gardn.et Champ.)幼苗土壤细菌多样性和群落组成的影响。结果表明:不同氮添加方式下,土壤细菌多样性和群落组成对氮添加的响应不同,并受到季节的影响。在雨季,LAN处理木荷土壤细菌多样性高于SAN处理;而旱季,LAN处理两种幼苗根系土壤细菌多样性均高于SAN处理。LAN处理条件下,马尾松土壤AcidobacteriaGp1相对丰度在旱季和雨季均显著高于SAN处理(P<0.05)。在雨季,马尾松LAN处理土壤Alphaproteobacteria相对丰度显著低于SAN处理(P<0.05),旱季没有显著差异(P>0.05)。氮添加能够提高木荷土壤细菌多样性,提高马尾松土壤变形菌门的相对丰度。旱季施氮还能够提高马尾松土壤Actinobacteria相对丰度,降低木荷土壤酸杆菌门的相对丰度。土壤细菌群落变化与土壤pH显著相关(P<0.05),木荷土壤细菌群落还受到土壤NH+4-N含量的显著影响(P<0.05)。     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu, Lake Mochou in Nanjing and Lake Taihu in Wuxi. Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted. The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g), while that of sediments in Lake Taihu was relatively low. After DNA purification, the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE. The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments. The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of microorganisms were identified in the sediment samples of both lakes. These results suggest that the sediment samples of these two city lakes (Xuanwu, Mochou) have similar microbial communities. However, the DGGE profiles of sediment samples in Lake Taihu were significantly different from these two lakes. Furthermore, the DGGE profiles of sediment samples in different locations in Lake Taihu were also different, suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou. The differences in microbial diversity may be caused by the different environmental conditions, such as redox potential, pH, and the concentrations of organic matters. Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced. The results of sequencing analysis indicate that five sequences shared 99%–100% homology with known sequences (Bacillus and Brevibacillus, uncultured bacteria), while the other two sequences shared 93%–96% homology with known sequences (Acinetobacter, and Bacillus). The study shows that the PCR-DGGE technique combined with sequence analysis is a feasible and efficient method for the determination of microbial communities in sediment samples. __________ Translated from Acta Ecologica Sinica, 2006, 26(11): 3610–3616 [译自: 生态学报]     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study,PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu,Lake Mochou in Nanjing and Lake Taihu in Wuxi.Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted.The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g),while that of sediments in Lake Taihu was relatively low.After DNA purification,the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE.The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments.The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of micro-organisms were identified in the sediment samples of both lakes.These results suggest that the sediment samples of these two city lakes (Xuanwu,Mochou) have similar microbial communities.However,the DGGE profiles of sediment samples in Lake Taihu were significantly differ-ent from these two lakes.Furthermore,the DGGE pro-files of sediment samples in different locations in Lake Taihu were also different,suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou.The differences in microbial diversity may be caused by the different environmental conditions,such as redox potential,pH,and the concentrations of organic matters.Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced.The results of sequencing analysis indicate that five sequences shared 99%-100% homology with known sequences (Bacillus and Brevibacillus,uncultured bacteria),while the other two sequences shared 93%-96% homology with known sequences (Acinetobacter,and Bacillus).The study shows that the PCR-DGGE tech-nique combined with sequence analysis is a feasible and efficient method for the determination of microbial com-munities in sediment samples.     

Seasonal dynamic changes in bacterial compositions in the Inner Mongolia desert steppe

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was employed to examine the seasonal dynamic changes in bacterial community composition in the Inner Mongolia desert steppe using specific primers F954 and R1369.Bright and reproducible bands were sequenced,and the phylogenic tree was constructed.The results show that the bacterial community composition changed between different seasons.The specific bands were different between the sampling sites with light and heavy levels of degraded grassland.Three main types of bacteria constituting the microbial community in the Inner Mongolia desert steppe belonged to the α,γ and δ-sub phyla of Proteobacteria,Bacteroidetes and Acidobacteria.The unculturable bacteria accounted for 69% of the whole bacterial community of the Inner Mongolia desert steppe.     

Seasonal dynamic changes in bacterial compositions in the Inner Mongolia desert steppe

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was employed to examine the seasonal dynamic changes in bacterial community composition in the Inner Mongolia desert steppe using specific primers F954 and R1369. Bright and reproducible bands were sequenced, and the phylogenic tree was constructed. The results show that the bacterial community composition changed between different seasons. The specific bands were different between the sampling sites with light and heavy levels of degraded grassland. Three main types of bacteria constituting the microbial community in the Inner Mongolia desert steppe belonged to the α, γ andgd-sub phyla of Proteobacteria, Bacteroidetes and Acidobacteria. The unculturable bacteria accounted for 69% of the whole bacterial community of the Inner Mongolia desert steppe.